The engagement of cell-surface receptors by extracellular ligands is a basic biological mechanism to initiate intracellular signaling cascades. Cross-reactivity of signaling receptors, for a spectrum of diverse ligands, is a property vital to the normal homeostasis of many physiologically-important systems. In vertebrates, gp130 is a shared cell-surface signaling receptor for a family of hematopoietic four-helix cytokines. Activation of gp130 results in a pleiotropic spectrum of both overlapping and unique cellular consequences that are mediated through JAK/STAT phosphorylation cascades. The activation of gp130 occurs through a sequential process initiated by cytokine recognition, followed by stepwise homo- and/or hetero-oligomerization into higher-order signaling assemblies. Hence, there is an inherent degeneracy in ligand recognition by gp130 which enables it to productively interact with a diverse family of cytokines. This degeneracy is coupled to the formation of higher order, cytokine-specific signaling assemblies. In this application we ask: 1- What is the molecular basis of the promiscuous recognition properties inherent in gp130's role as a shared receptor ?, and 2- What are the architectures of the extracellular higher-order gp130-cytokine signaling assemblies that result in activation of this receptor? To address these questions, we combine structural analysis of large multimeric receptor complexes with detailed biochemical dissection of the energetics driving these interactions within the context of the larger assembly. We are focusing on interleukin-6, viral interleukin-6 and CNTF as structurally diverse cytokines which utilize common gp130 binding sites to assemble unique homo- and hetero-oligomeric signaling assemblies. We will express engineered, recombinant forms of these cytokines and their receptor ectodomains, which will then be utilized to reconstitute the assembly pathways, carry out thermodynamic and kinetic measurements, and determine the three-dimensional structures of the complexes by x-ray crystallography. Lastly, we will utilize molecular and computational protein engineering approaches to probe the structural energetics of the receptor/cytokine interfaces, and design variant proteins with novel binding and activation properties that may be of therapeutic value. The long-term goal of this project is to begin to bridge the gap linking extracellular cytokine recognition to the initiation of signaling events.